EPR-Based Distance Measurements on Metal Nitroxide Model Complexes as well as EPR Spectroscopic Characterization of [Si₂]-Radicals and Titanocene Complexes

Abstract

Metalloproteins are a class of proteins which contain metal ions in their active site. If these metal ions carry unpaired electrons, EPR spectroscopy can be used to identify and characterize these ions or even localize them within the protein backbone. This work describes the synthesis of chemical compounds containing paramagnetic metal ions and nitroxyl radicals and their application as model compounds for EPR distance measurements in metalloproteins or other metal containing biomolecules. In addition, the detection, identification, and characterization of reactive, paramagnetic substances using EPR spectroscopy is described. Chapter 1 gives a brief introduction into both fields, emphasizing the potential and importance of EPR spectroscopy in certain fields of structural biology as well as the versatility of EPR spectroscopy as a tool for the investigation of the electronic and geometric structure of small chemical compounds. Chapter 2 introduces the basic theoretical background of EPR spectroscopy, which is needed to understand the following chapters describing the experimental results. In chapters 3 and 4, the synthesis and crystallization of nitroxide substituted terpyridine ligands and their geometrical structure are described. The terpyridine and the nitroxide subunits are separated by phenylene and ethynylene building blocks. It is found, that the phenylene-ethynylene spacers are sufficiently rigid to yield predictable distances between the terpyridine nitrogen atoms and the nitroxide substituent. Chapter 5 describes the synthesis, and the full crystallographic and spectroscopic characterization of bis(terpyridine) copper bis(tetraphenylborate). It is shown that the coordination sphere is highly plastic and exhibits temperature dependent, dynamic and static Jahn-Teller distortions. These distortions are influenced by the choice of the solvent system and affect the EPR parameters of the copper ion, which is of importance for the EPR distance measurements described in chapter 6. There, the synthesis of homo- and nominally heteroleptic copper complexes of the terpyridine ligand introduced in chapter 4 and unsubstituted terpyridine is described. The resulting complexes are then used as model systems for EPR distance measurements. Using PELDOR, it is found that the obtained time traces are distorted by orientation selection and multi-spin effects. The RIDME experiment on the other hand is not subject to these effects and provides better sensitivity. Additionally, both experiments are used to investigate the immediate coordination sphere of the copper center. It is shown that one terpyridine ligand is removed from the central copper ion in acidic solvent systems. In chapter 7, the manganese(II) analogue of the homoleptic copper complex is used as model system for EPR distance measurements involving high-spin ions. The most striking difference between the copper and the manganese complex is the high-spin (S = 5/2) state of the manganese ion. It is shown that orientation selection also plays a role in this system, albeit the selectivity is less pronounced than in the case of the copper system. In terms of sensitivity, RIDME is superior to PELDOR in the manganese system as well. However, RIDME suffers from the occurrence of intense artifact peaks due to higher harmonics of the dipolar frequency. The degree of ligand dissociation is estimated using PELDOR. The studies on the metal-nitroxide model systems show up difficulties in EPR distance determinations that involve metal centers and possible ways to overcome those difficulties. The obtained results will be conveyed to biological systems in future studies, where one possible application would be the introduction of manganese(II) ions into RNA molecules and the subsequent use of these ions as spin label.<br /> The final three chapters describe the detection and characterization of reactive paramagnetic substances in their natural solvent environment. In chapter 8, the structure of a paramagnetic, N-heterocyclic carbene substituted disilicon radical cation containing a Si-Si multiple bond in solution is investigated. Using the 29Si satellites it is found that the radical is less symmetric than its Lewis formula would suggest. In chapter 9, the structure of an "Si2H" radical stabilized by N-heterocyclic carbenes is investigated in liquid solution. The structure of the nitrogen hyperfine coupling suggests that the hydrogen atom is localized on one silicon atom. Chapter 10 focuses on catalytically active titanocene species used for epoxide hydrosilylation. Two titanocene hydride species are identified as hydrogen atom donors. Furthermore, a titanocene alkoxide species which occurs as resting state of the catalytic cycle is identified using deuteration experiments and ENDOR spectroscopy. Taken together, chapters 8 - 10 demonstrate that EPR spectroscopy can be used to obtain detailed pictures of the electronic and geometric structure of reactive paramagnetic compounds in liquid and frozen solvent matrices.